SUMMARY/ABSTRACT Celiac disease (CeD) affects approximately 1% of Americans. While may patients respond well to a gluten-free diet, objective measures of dietary compliance are lacking while discriminating between the lack of compliance and refractory disease has major implications for the patient?s risk of developing cancer. High- dimensional mass cytometry (cytometry by time-of-flight; CyTOF) combines flow cytometry with mass spectrometry, enabling the simultaneous quantification of more than 40 surface and/or intracellular markers expressed by subsets of immune/inflammatory cells. This cutting-edge technology now allows us to perform deep immunophenotyping of the surface (e.g. CD molecules) and intracellular molecular profile (cytokines, transcription factors) of the cells within the inflamed intestine with unprecedented resolution. Using computational tools designed to analyze complex, single-cell datasets, phenotypic signatures can be ascribed to cohorts reflecting different conditions or stages of disease progression We have designed a study to apply mass cytometry to determine the phenotype of cells in the duodenal mucosa of human controls as well as patients with celiac disease including: newly diagnosed not on a gluten- free diet (NEW); patients on a gluten-free diet (GF); and patients with refractory celiac disease (RCD). The data obtained thus far identify that the mucosa from patients with RCD-II contains T cells expressing a distinct array of homing molecules. These data lead to our overall hypothesis that immunophenotyping host immune/inflammatory cells will identify subsets that serve as a biomarker to stratify cohorts of patients with different presentations of celiac disease. This hypothesis will be addressed in the following Specific Aims: Aim 1: Define the immunophenotype in human subjects with or without celiac disease. Aim 2: Validate markers identified by mass cytometry in refractory celiac disease. This exploratory project extends our preliminary findings to apply the technology to an increased number of subjects; to extrapolate findings from mass cytometry in tissue to peripheral blood; to validate the unique phenotypes that are detected; and to evaluate the association of the newly-defined subsets with the cohorts of patients with different presentations of celiac disease. These findings may provide new diagnostic tools to distinguish refractory celiac disease from patients failing dietary compliance or other presentations of the condition. In addition, subsets of immune cells that are enriched in RCD may identify potential therapeutic targets to aid in treating the condition and preventing the progression to cancer.